From owner-biotechniques@net.bio.net Wed May 07 23:00:00 1997
Path: biosci!biosci!not-for-mail
From: wfischer@sunflower.bio.indiana.edu (Will Fischer)
Newsgroups: bionet.journals.letters.biotechniques
Subject: Re: protoplast buffer and lysis buffer
Date: 8 May 1997 06:13:12 -0700
Organization: Biology, Indiana University - Bloomington
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Robyn Puffenbarger (rpuffenbarg@Gems.VCU.EDU) wrote:
: 
: I have the protocol for the "in-well-lysis" procedure, but have lost the 
: recipies for the protoplast buffer and the lysis buffer.  This simple 
: technique lets you quickly see the difference in recombinant plasmids 
: without a 'miniprep.'  

Protoplasting buffer:

30 mM	 Tris-HCl pH 8.0
 5 mM	 Na2EDTA
50 mM	 NaCl
20 %	 sucrose
50 ug/ml RNase A
50 ug/ml lysozyme

(I also add 1 mg/ml Orange G)

Lysis buffer:

89 mM	 Tris
89 mM	 boric acid
 2.5 mM	 Na2EDTA
 2 %	 SDS
 5 %	 sucrose
 0.04 %	 bromophenol blue


Reference:  Sekar, V. (1987) BioTechniques 5:11-13

Alternative technique:

Pick colonies for PCR with vector primers (protocol available at
http://www.bio.indiana.edu/~wfischer/protocol_colony-PCR-screen.html).

This allows preparation of sequencing template as part of the screening step!
-- 
____________________________________________________________
Will Fischer			

Biology Department             		wfischer@indiana.edu
Jordan Hall                   		http://www.bio.indiana.edu/~wfischer
Indiana University            		Lab:    812-855-2549
Bloomington, Indiana 47405 USA		FAX:    812-855-6705

.